The subject of the present invention is compounds having the property of modifying serotoninergic transmission as well as the diagnostic and therapeutic applications of these compounds.
The cerebral serotoninergic system is a cerebral and peripheral neurotransmission system which uses serotonin or 5-hydroxytryptamine (5-HT) which was discovered during the course of 1949/50.
At the central level, this system is special in that it is extremely centralized (all the cellular bodies are located in the posterior region of the brain, the raphe) and where it projects into practically all the regions of the brain. It is characterized by an extremely high level of varicosity (or equivalent of neuronal ends) along the axons; this arrangement multiplies its points of interaction with the other cerebral neuronal systems. Furthermore, a very large part of this varicosity does not exhibit any synaptic profile, that is to say that the 5-HT released by these xe2x80x9cendsxe2x80x9d will diffuse and reach the targets (receptors) located at a certain distance; this mechanism requires the use of special receptors having a high affinity for the amine. This structural arrangement is quite favourable for a function of controlling the entire neuronal cells of the brain, and consequently for an essential role in maintaining the homeostasis of the central nervous system.
To exert this control, the 5-HT system uses a large variety of specific receptors. Several families are indeed involved some of which are well characterized (5-HT1, 5-HT2, 5-HT3, 5-HT4) and others identified more recently are still not well known (5-HT5, 5-HT6, 5-HT7). Some of them are represented by numerous subtypes; such is the case in particular for the 5-HT1xe2x88x92 family, characterized by a nanomolar affinity of 5-HT for these sites, and which comprises the subtypes 5-HT1A,B,D,E,F.
The 5-HT1B and 5-HT1D type receptors are located on the serotoninergic and non-serotoninergic ends where they modulate the release of the neurotransmitter contained in these ends, in particular they can regulate the release of 5-HT itself (autoreceptors) and play, in this case, a decisive role in the activity of the serotoninergic neurotransmission.
From a fundamental point of view, the serotoninergic system is involved in numerous physiological functions and in numerous pathologies, and especially in depressive syndrome.
Depression is a psychiatric disorder which is still poorly known in spite of numerous studies which have been devoted to it. However, it is now quite clear that most, if not all, depressions involve the serotoninergic system and, especially, it is generally accepted that there is a relationship between a serotoninergic deficiency and suicide.
In addition, antidepressant drugs interact, for a large number of them, with the serotoninergic system and the most recent, which are the most effective and which possess the least undesirable effects, are most often of the xe2x80x9cserotoninergicxe2x80x9d type. These substances all result, by various mechanisms, in facilitating serotoninergic transmission (inhibitors of monoamine oxidases, inhibitors of recapture, 5-HT1A agonists). However, on the one hand, their efficacy is limited and, on the other hand, they still possess, for many of them, substantial side effects, and finally, their delayed action is substantial ( greater than 2 weeks).
Recent results in the literature and from the inventors indicate that the 5-HT1B/1D receptors might play a major role in the depressive syndrome and in the mechanism of action of antidepressants. It has indeed been shown that they not only modulate the liberation of acetylcholine, but they also modulate that of 5-HT itself and thereby effectively regulate the serotoninergic transmission of which the importance in the depressive syndrome is known. Furthermore, they are the target of antidepressants which interact probably in a noncompetitive manner; in other words, the antidepressants are thought to recognize a separate site of the 5-HT1B/1D receptors, but closely interactive with the latter.
The work of the inventors has allowed the identification of a cerebral endogenous compound which recognizes a receptor site capable of interacting with the functioning of certain 5-HT receptors, in particular the 5-HT1B/1D receptors which modulate the release of 5-HT itself.
The cerebral endogenous compound identified is a tetrapeptide of sequence:
Leu-Ser-Ala-Leu (sequence LSAL SEQ ID NO:4).
The inventors also tested the activity of peptides comprising part of the LSAL sequence or the modified LSAL sequence and showed that these compounds still had a good activity on the 5-HT1B/1D receptors. In general, a modification (for example an acetylation) of the NH2-terminal end leads to a compound having a lower ligand activity on the 5-HT receptor, whereas modifications of the COOH end make it possible to obtain a peptide which still has a substantial activity.
For example, the addition of the sequence Gly-Gly-Gly-Tyr to the COOH end of the LSAL sequence makes it possible to obtain a compound still having a substantial activity. However, the addition of the same sequence, but whose Tyr residue is iodinated, leads to a compound no longer having a ligand activity for the 5-HT receptor, probably because of the steric hindrance due to the large size of the iodine atoms which prevent the binding of the peptide thus modified to its receptor.
The subject of the invention is thus a compound of peptide sequence
X Leu Y
in which X represents H (SEQ ID NO:1) or Ala (SEQ ID NO:2) or Leu-Ser-Ala (SEQ ID NO:3), Y represents OR, or a peptide sequence having from 1 to 10 amino acids, whose carboxy-terminal end is amidated by an NH2 group or esterified by a substituted or unsubstituted hydroxycarbyloxy residue, with the proviso that X and Y do not simultaneously represent H and OH, respectively as well as the corresponding compounds in which the peptide bonding group xe2x80x94COxe2x80x94NHxe2x80x94 is replaced by a bonding group resistant to the enzymatic degradation of proteases, or in which the peptide backbone comprises one or more intercalated groups making the peptide bond resistant to enzymatic degradation.
Thus, the peptide bonding group (xe2x80x94COxe2x80x94NHxe2x80x94) is advantageously replaced by a bonding group xe2x80x94COxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94CR1R2xe2x80x94CR3R4xe2x80x94, xe2x80x94COxe2x80x94CR1R2xe2x80x94, R1, R2, R3 and R4, which are identical or different, representing H or a C1-C6 alkyl group, especially a methyl group, and Rxe2x80x2 representing a C1-C6 alkyl group.
Among the groups which can be intercalated into the peptide backbone, the following groups may be mentioned xe2x80x94CR1R2xe2x80x94, xe2x80x94NR1xe2x80x94 and xe2x80x94Oxe2x80x94, R1 and R2 being defined as above.
Hydroxycarbyloxy residue is understood to mean especially an alkyloxy, alkenyloxy or alkynyloxy group, with a linear or branched chain, having from 1 to 10 carbon atoms, unsubstituted or substituted by one or more groups selected from OH, NH2, NO2, Cl, Br, F, CF3 for example the group OCH3, OC2H5, OCHOHxe2x80x94CH3, OCHOHxe2x80x94CH2OH, OCH2CH2NH2, OC3H7 and the like.
The subject of the invention is more particularly a compound of the following peptide sequence:
Leu-Ser-Ala-Leu-Z (SEQ ID NO:3)
in which Z represents OH, NH2 a substituted or unsubstituted hydrocarbyloxy residue, or a peptide sequence having from 1 to 10 amino acids, as well as the corresponding compounds in which the peptide bond is replaced by a bond resistant to the enzymatic degradation by proteases or in which the peptide backbone comprises one or more intercalated groups making the peptide bond resistant to enzymatic degradation.
The nature of the amino acids of Z is unimportant, the limit being that when modified amino acids are involved (not encoded genetically), these are not substituted by a bulky group (having a size greater than that of the radius of the iodine atom).
The compounds according to the invention comprise especially the peptides of the following sequence:
Ala-Leu;
Leu-Ser;
Ala-Leu-Ser;
Leu-Ser-Ala-Leu (SEQ ID NO:4);
Leu-Ser-Ala-Leu-OCH3 (SEQ ID NO:4);
Leu-Ser-Ala-Leu-NH2 (SEQ ID NO:4);
Leu-Ser-Ala-Leu-Gly-Gly-Gly-Tyr (SEQ ID NO:5);
as well as the analogous compounds in which the peptide bond is replaced by a bond as defined above; or whose backbone comprises an intercalated group as defined above.
The most active compounds are those whose constituent amino acids are in the L form, that is to say the natural form for the peptide structures defined above.
The compounds according to the invention are prepared by extraction from freeze-dried brain as described below or more advantageously by conventional peptide synthesis, for example by the solid-phase method of MERRIFIELD when they are exclusively of peptide nature, or alternatively by modification of the terminal groups of the amino acids and condensation of the residues thus obtained, the reactive functional groups other than those engaged in the bonding between the successive residues having been previously protected with the aid of protecting groups well known to a person skilled in the art.
According to one example of preparation of a peptide according to the invention, the first residue of the COOH terminal end whose amine functional group is protected by a tert-butyloxycarbonyl group is attached to a resin, via its carboxyl group, then, after deprotecting the amine functional group by washing the resin with trifluoroacetic acid in dichloromethane, the second amino acid residue whose amino functional group is protected as above is coupled in dimethylformamide; the amino acid residues which will constitute the peptide portion according to the invention are thus attached one after the other. After deprotection, the amine functional group of the N-terminal residue can be acetylated by the action of excess acetic anhydride in the presence of diisopropylethylamine.
The reactive side chains of the amino acids can be protected for example by a benzyl group, for the hydroxyl side chains.
After removing the protecting group, the peptide according to the invention is removed from the solid support, for example with hydrofluoric acid. The crude product is freeze-dried and subjected to liquid chromatography at moderate pressure, which makes it possible to obtain a practically pure product; the latter is then characterized by high-performance liquid chromatography as well as by analysing its amino acid composition and by mass spectrometry.
The peptides characterized possess the property of modifying serotoninergic transmission through its interactions with the autoreceptors (and also the hetero-receptors) and play a key role in various psychiatric pathologies where the 5-HT system is notoriously involved (stress, anxiety, depression, compulsive obsessions, appetite disorders, sleep disorders, behavioural disorders, aggressiveness and the like).
The subject of the invention is also a compound capable of acting as ligand for the receptor to which the endogenous peptide of sequence Lou-Ser-Ala-Leu (LSAL: SEQ ID NO:4) attaches, characterized in that it comprises a group whose spatial structure is substantially identical to that of a peptide of sequence
X-Leu-Y
in which X and Y are as defined above, X and Y not simultaneously representing H and OR, respectively.
Such a compound may be an agonist or an antagonist of the peptide LSAL.
xe2x80x9cSubstantially identical spatialxe2x80x9d structure is understood to mean for the purposes of the present invention that the mean position of the atoms forming the group which binds to the receptor only differ at most by 5%, and preferably at most by 2% from the mean position of the atoms forming the corresponding peptide.
Among the preferred compounds, there may be mentioned those comprising a group whose spatial structure is substantially identical to the following di-, tri- and tetrapeptides:
Ala-Leu
Leu-Ser
Ala-Leu-Ser
Leu-Ser-Ala-Leu (SEQ ID NO:4).
The compounds according to the invention are produced from the spatial structures(s) of the peptides according to the invention by computer modelling and conventional chemical synthesis using the structure obtained after modelling, as described in J. Med. Chem., vol. 37, No. 9, pp. 1233 to 1251 and J. Med. Chem., vol. 37, No. 10, pp. 1385-1401.
Advantageously, these compounds are produced, in addition, based on the spatial structure of known antidepressant molecules active on this same site.
The modelling technique makes it possible in particular to define and characterize the structure of molecules not affecting the sites of transport of amines (contrary to most currently known antidepressants) but affecting the unique allosteric site carried by the 5-HT1B/D receptors.
The subject of the invention is also a therapeutic composition comprising a compound as defined above, capable of crossing the haematoencephalic barrier.
To cross the hematoencephalic barrier, the peptide is preferably administered in the form of a prodrug, especially of the glycosyl phosphotriester type, in which the peptide is bound for example through the leucine to the phosphate group, as described in J. MED. CHEM. 92, Vol. 35, p. 30-39.
Such a prodrug, as well as in general a precursor molecule capable of releasing in vivo a compound according to the invention, constitutes another subject of the invention.
The therapeutic composition is advantageously used in pathologies in which the serotoninergic system is involved, especially in the pathologies linked to a deficiency of the serotoninergic transmission.
Thus, the composition according to the invention can be used in particular in the treatment of depression, but also in all the indications currently covered by the antidepressants, such as compulsive obsessional disorders, generalized anxiety, panic attack, appetite disorders, sleep disorders, impulsivity, sexual disorders, aggressivity, and more generally those for the compounds which facilitate serotoninergic transmission.
The composition according to the invention is advantageously provided in an injectable or oral form or in any other known pharmaceutical form.
The effective therapeutic dose is easily determined by a person skilled in the art depending on the nature and the seriousness of the pathology to be treated, as well as the weight and age of the patient. The compounds according to the invention are not toxic. They are added to a pharmaceutically acceptable vehicle or to an excipient well known to a person skilled in the art.
The subject of the invention is also a process for the in vitro diagnosis of a condition linked to the serotoninergic system in a patient, characterized in that a peptide of sequence
Leu-Ser-Ala-Leu (SEQ ID NO:4)
is assayed in a biological fluid from the patient.
The assay is carried out especially by an immunological method comprising the bringing of the biological sample into contact with an antibody specifically directed against a peptide according to the invention and the detection of the antigen-antibody complex thus formed.
These processes may be based on a radio-imunological method of the RIA, RIPA or IRMA type, or on an immunoenzymatic method, for example of the ELISA type.
The biological fluid may be blood, urine or the spinal fluid.
The antibodies may be monoclonal or polyclonal and constitute, as well as their Fab, Fabxe2x80x2, F(abxe2x80x2)2 and Fc fragments, another subject of the invention.
The antibodies are obtained by coupling a peptide according to the invention to an immunogenic carrier peptide or protein.
The subject of the invention is also a diagnostic kit for assaying a peptide according to the invention in a body fluid from a patient in whom a pathology linked to a deficiency in serotoninergic transmission, especially a masked depression, is suspected, comprising at least one antibody as defined above and optionally the reagents for carrying out the assay.
The peptides of the invention have proved to be inactive on the site of recapture of 5-HT.
The existence of a cerebral endogenous factor not active on the site of recapture constitutes a reference standard in terms of activity and thus makes it possible to envisage a process for the selection of antidepressant substances, consisting in discriminating between the recapture effect and the interaction effect on the 5-HT1B/1D site.
This process applies to known antidepressants and also allows the development of new drugs.
In practice, the discrimination between the recapture effect and the effect on the 5-HT1B/1D site can be carried out by performing tests of inhibition of binding between, on the one hand, increasing quantities of antidepressants and the capture of 5-HT in the synaptosomes, the 5-HT being radiolabelled and, on the other hand, by performing tests of the same type on the 5-HT1B/1D receptor by varying the quantity of antidepressants in the presence of a radiolabelled ligand specific for these receptors, and by determining the respective Ki values or more generally the IC50 values.
The choice of antidepressants having a selective activity on the 5-HT1B/1D site will be dictated by the substances having a high Ki or a low IC50 in the recapture test and conversely a low Ki or a high IC50 in the test of inhibition of binding to the peptide recognition site of the 5-HT1B/1D. The radiolabelled (tritium) peptide can also and more easily be used to identify and characterize the specific site for recognition of the peptide, and thus directly to study the capacities of various substances to displace (competition) this peptide by measuring their Ki and by comparing the latter with the Ki values obtained for these same substances to inhibit the capture of 5-HT (or displace a recapture inhibitor, 3H-paroxetine for example).
Another subject of the invention also consists in a process for the detection of ligands of the 5-HT1B/1D receptor site for the peptide LSAL, characterized in that there are carried out the steps consisting in:
bringing a molecule or a mixture containing various molecules, optionally not identified, into contact with a recombinant cell expressing the 5-HT1B/1D receptor at its surface, in the presence of a compound according to the invention under conditions allowing the interaction between the 5-HT1B/1D receptor and the said molecule(s), in the case where the latter might have an affinity for the 5-HT1B/1D receptor;
detecting the quantity of the said compound according to the invention bound to the receptor;
deducing therefrom the possible attachment of the said molecule(s).
This process can be used for the detection of agonists or antagonists of the peptide LSAL.
Another subject of the invention relates to a process for the detection of modulators (antagonists or agonists) of the 5-HT1B/1D receptor site for the peptide LSAL characterized in that there are carried out the steps consisting in:
bringing a molecule or a mixture containing various molecules, optionally not identified, into contact with a recombinant cell expressing the 5-HT1B/1D receptor at its surface, in the presence of a compound according to the invention under conditions allowing the interaction between the 5-HT1B/1D receptor and the said compound according to the invention;
detecting the molecules capable of mimicking or antagonizing the activity of the compound according to the invention on the said receptor.
Ligands obtained according to a process as described above can be used as active ingredient of a therapeutic composition useful in the treatment of psychiatric disorders such as depression, circulatory disorders such as migraine, or immunological disorders linked to the 5-HT1B/1D receptors.
The compounds according to the invention are also useful as diagnostic tools for labelling the 5-HT1B/1D receptors, and more particularly the endogenous peptide binding site on these receptors.
To this end, a diagnostic composition comprising a radiolabelled compound according to the invention is advantageously used in an imaging technique using a positon emission tomography scanner.